CA2402321A1 - Dna joining method - Google Patents
Dna joining method Download PDFInfo
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- CA2402321A1 CA2402321A1 CA002402321A CA2402321A CA2402321A1 CA 2402321 A1 CA2402321 A1 CA 2402321A1 CA 002402321 A CA002402321 A CA 002402321A CA 2402321 A CA2402321 A CA 2402321A CA 2402321 A1 CA2402321 A1 CA 2402321A1
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- dna
- dna molecule
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Abstract
The present invention provides a method to directionally clone any linear template DNA molecule into any linearized vector. The vector ends may be generated from any restriction enzyme cleavage. The method does not require a ligation step nor the use of carefully controlled conditions as is required with methods involving specific exonucleases alone. It has been determined that specific DNA polymerases are able to efficiently join one or more linear DNA molecules sharing ends with appropriate complementation.
Claims (43)
1. A method of joining two linear DNA molecules comprising the step of:
- providing a first linear DNA molecule having a first end and a second end;
- providing a second linear DNA molecule having a first end and a second end;
wherein the first linear DNA molecule has a sequence of nucleotides at either the first end or the second end that is complementary to a sequence of nucleotides at either the first end or the second end of the second linear DNA
molecule; and - incubating the two linear DNA molecules in the presence of a DNA
polymerise under conditions whereby the two linear DNA molecules are joined.
- providing a first linear DNA molecule having a first end and a second end;
- providing a second linear DNA molecule having a first end and a second end;
wherein the first linear DNA molecule has a sequence of nucleotides at either the first end or the second end that is complementary to a sequence of nucleotides at either the first end or the second end of the second linear DNA
molecule; and - incubating the two linear DNA molecules in the presence of a DNA
polymerise under conditions whereby the two linear DNA molecules are joined.
2. A method of joining three linear DNA molecules comprising:
- providing a first linear DNA molecule having a first end and a second end;
- providing a second linear DNA molecule having a first end and a second end;
- providing a third linear DNA molecule having a first end and a second end;
wherein the first linear DNA molecule has a sequence of nucleotides at the first end that is complementary to a sequence of nucleotides at either the first or second end of the second linear DNA molecule and wherein the first linear DNA
molecule has a sequence of nucleotides at the second end that is complementay to a sequence of nucleotides at either the first or second end of the third linear DNA
molecule, and - incubating the three linear DNA molecules in the presence of a DNA
polymerise under conditions whereby the three linear DNA molecules are joined.
- providing a first linear DNA molecule having a first end and a second end;
- providing a second linear DNA molecule having a first end and a second end;
- providing a third linear DNA molecule having a first end and a second end;
wherein the first linear DNA molecule has a sequence of nucleotides at the first end that is complementary to a sequence of nucleotides at either the first or second end of the second linear DNA molecule and wherein the first linear DNA
molecule has a sequence of nucleotides at the second end that is complementay to a sequence of nucleotides at either the first or second end of the third linear DNA
molecule, and - incubating the three linear DNA molecules in the presence of a DNA
polymerise under conditions whereby the three linear DNA molecules are joined.
3. A method of joining two or more linear DNA molecules comprising the steps of:
- providing two or more linear DNA molecules, each having an x and x' strand having opposite polarities, wherein the 5' end of the x' strand of one linear DNA molecule has a sequence of nucleotides that is complementary°
to the 5' end of the x strand of the linear DNA molecule to which it is to be joined; and - incubating the two or more DNA molecules in the presence of a DNA
polymerase under conditions whereby the two or more linear DNA
molecules are joined;
wherein the DNA polymerase has intrinsic exonuclease activity and joins the DNA molecules.
- providing two or more linear DNA molecules, each having an x and x' strand having opposite polarities, wherein the 5' end of the x' strand of one linear DNA molecule has a sequence of nucleotides that is complementary°
to the 5' end of the x strand of the linear DNA molecule to which it is to be joined; and - incubating the two or more DNA molecules in the presence of a DNA
polymerase under conditions whereby the two or more linear DNA
molecules are joined;
wherein the DNA polymerase has intrinsic exonuclease activity and joins the DNA molecules.
4. The method according to claim 1, 2 or 3, wherein the DNA polymerase has 3'-
5'-exonuclease activity.
5. The method according to claim 4, wherein the DNA polymerase comprises a polymerase selected from the group consisting of vaccinia virus DNA
polymerase, T4 DNA polymerase and the Klenow fragment of E. coli DNA
polymerase I.
5. The method according to claim 4, wherein the DNA polymerase comprises a polymerase selected from the group consisting of vaccinia virus DNA
polymerase, T4 DNA polymerase and the Klenow fragment of E. coli DNA
polymerase I.
6. The method according to claim 5, wherein the DNA polymerase comprises vaccinia virus DNA polymerase.
7. The method according to any one of claims 1-6, wherein the two or more linear DNA molecules are incubated in the presence of a DNA polymerase and a stimulatory factor.
8. The method according to claim 7, wherein the stimulatory factor is a single strand DNA binding protein.
9. The method according to claim 8, wherein the single strand DNA binding protein is vaccinia single strand DNA binding protein.
10. The method according to any of claims 1 to 9, wherein the length of the sequence of complementary nucleotides on each linear DNA molecule is between about 5 and about 100 nucleotides.
11. The method according to any one of claims 1 to 9, wherein the length of the sequence of complementary nucleotides on each linear DNA molecule is between about 8 and about 50 nucleotides.
12. The method according to any one of claima 1 to 9, wherein the length of the sequence of complementary nucleotides on each linear DNA molecule is between about 10 and about 35 nucleotides.
13. A method of circularizing linear DNA molecules comprising:
- PCR amplifying a linear DNA molecule in the presence of primers that will introduce substantially complementary nucleic acid sequences onto each end of the linear DNA molecule; and - incubating the PCR amplified linear DNA molecule in the presence of a DNA polymerase under conditions where the linear DNA molecule is circularized;
wherein the DNA polymerase has intrinsic exonuclease activity and circularizes the DNA molecule.
- PCR amplifying a linear DNA molecule in the presence of primers that will introduce substantially complementary nucleic acid sequences onto each end of the linear DNA molecule; and - incubating the PCR amplified linear DNA molecule in the presence of a DNA polymerase under conditions where the linear DNA molecule is circularized;
wherein the DNA polymerase has intrinsic exonuclease activity and circularizes the DNA molecule.
14. A method of constructing a recombinant molecule comprising the steps of:
- providing a linearized vector DNA molecule and a template DNA
molecule, each having a first and a second end;
providing a first primer DNA molecule having a 5' end that comprises nucleotide sequences that will incorporate nucleotide sequences that are complementary to the first end of the linearized vector molecule onto the first end of the template DNA molecule and a 3' end that hybridizes to a suitable location on the first end of the template DNA molecule;
- providing a second primer DNA molecule having a 5' end that comprises nucleotide sequences that will incorporate nucleotide sequences that are complementary to the second end of the linearized vector molecule onto the second end of the template DNA molecule and a 3' end that hybridizes to a suitable location on the second end of the template DNA molecule;
- amplifying the template DNA molecule using the polymerase chain reaction with the first and second primers to provide a PCR amplified product; and - incubating the PCR amplified product with the linearized vector DNA
molecule in the presence of a DNA polymerase under conditions to generate a recombinant DNA molecule;
wherein the DNA polymerase has intrinsic exonuclease activity and joins the DNA molecules.
- providing a linearized vector DNA molecule and a template DNA
molecule, each having a first and a second end;
providing a first primer DNA molecule having a 5' end that comprises nucleotide sequences that will incorporate nucleotide sequences that are complementary to the first end of the linearized vector molecule onto the first end of the template DNA molecule and a 3' end that hybridizes to a suitable location on the first end of the template DNA molecule;
- providing a second primer DNA molecule having a 5' end that comprises nucleotide sequences that will incorporate nucleotide sequences that are complementary to the second end of the linearized vector molecule onto the second end of the template DNA molecule and a 3' end that hybridizes to a suitable location on the second end of the template DNA molecule;
- amplifying the template DNA molecule using the polymerase chain reaction with the first and second primers to provide a PCR amplified product; and - incubating the PCR amplified product with the linearized vector DNA
molecule in the presence of a DNA polymerase under conditions to generate a recombinant DNA molecule;
wherein the DNA polymerase has intrinsic exonuclease activity and joins the DNA molecules.
15. The method according to claim 14, wherein the PCR amplified product and the linearized vector DNA molecule are incubated in the presence of a DNA
polymerase having 3'-5' exonuclease activity.
polymerase having 3'-5' exonuclease activity.
16. The method according to claim 15, wherein the DNA polymerase comprises a polymerase selected from the group consisting of vaccinia virus DNA
polymerase, T4 DNA polymerase and the Klenow fragment of E. coli DNA
polymerase I.
polymerase, T4 DNA polymerase and the Klenow fragment of E. coli DNA
polymerase I.
17. The method according to claim 16, wherein the DNA polymerase comprises vaccinia virus DNA polymerase.
18. The method according to any one of claims 15-17, wherein the PCR amplified product and the linearized vector DNA molecule are incubated in the presence of a DNA polymerase and a stimulatory factor.
19. The method according to claim 18, wherein the stimulatory factor is a single strand DNA binding protein.
20. The method according to claim 19, wherein the single strand DNA binding protein is vaccinia single strand DNA binding protein.
21. The method according to any one of claims 15-20, wherein the sequence of complementary nucleotides on each linear DNA molecule is between about 5 and about 100 nucleotides.
22. The method according to any one of claims 15 to 20. wherein the sequence of complementary nucleotides on each linear DNA molecule is between about 8 and about 50 nucleotides.
23. The method according to any one of claims 15 to 20, wherein the sequence of complementary nucleotide DNA molecule is between about 10 and about 35 nucleotides.
24. The method according to any of claims 15-23, wherein the vector is capable of transfecting a prokaryotic or eukaryotic host cell.
25. The method according to claim 24, wherein the vector comprises a vector selected from the group consisting of plasmids, cosmids, phage and BACs.
26. The method according to claim 25, wherein the vector comprises a plasmid.
27. The method according to claim 26, wherein the vector comprises pDW101.
28. A method of constructing a recombinant DNA molecule comprising:
- providing a linearized vector DNA molecule;
- providing a linear insert DNA molecule having ends that are substantially complementary to the ends of the linearized vector DNA molecule to which they are to be joined; and - incubating the linearized vector DNA molecule and linear insert DNA
molecule in the presence of a DNA polymerase under conditions where the two DNA molecules are joined;
wherein the DNA polymerase has intrinsic exonuclease activity and joins the DNA molecules.
- providing a linearized vector DNA molecule;
- providing a linear insert DNA molecule having ends that are substantially complementary to the ends of the linearized vector DNA molecule to which they are to be joined; and - incubating the linearized vector DNA molecule and linear insert DNA
molecule in the presence of a DNA polymerase under conditions where the two DNA molecules are joined;
wherein the DNA polymerase has intrinsic exonuclease activity and joins the DNA molecules.
29. The method according to claim 28, wherein the linear insert DNA molecule is obtained by linearization of a circular double strand DNA molecule by restriction enzyme digestion, with subsequent attachment of synthetic oligomer linkers onto the ends; or by secondary digestion of an already linear DNA molecule with restriction enzymes, followed by addition of adapter/linkers onto the ends; or by annealing of two appropriately designed synthetic single strand DNA oligomers to form an intact duplex DNA
molecule.
molecule.
30. A method of producing a recombinant DNA product comprising the steps of:
- providing a linearized vector DNA molecule and a template DNA
molecule, each having a first and a second end;
- providing a first primer DNA molecule having a 5' end that comprises nucleotide sequences that will incorporate nucleotide sequences that are complementary to the first end of the linearized vector molecule onto the first end of the template DNA molecule and a 3' end that hybridizes to a suitable location on the first end of the template DNA molecule;
- providing a second primer DNA molecule having a 5' end that comprises nucleotide sequences that will incorporate nucleotide sequences that are complementary to the second end of the linearized vector molecule onto the second end of the template DNA molecule and a 3' end that hybridizes to a suitable location on the second end of the template DNA molecule;
- amplifying the template DNA molecule using the polymerase chain reaction with the first and second primers to provide a PCR amplified product;
incubating the PCR amplified product with the linearized vector DNA
molecule in the presence of a DNA polymerase under conditions to generate a recombinant DNA molecule;
- transforming the recombinant DNA molecule into a host cell; and - isolating the recombinant DNA product;
wherein the DNA polymerase has intrinsic exonuclease activity and joins the DNA molecules.
- providing a linearized vector DNA molecule and a template DNA
molecule, each having a first and a second end;
- providing a first primer DNA molecule having a 5' end that comprises nucleotide sequences that will incorporate nucleotide sequences that are complementary to the first end of the linearized vector molecule onto the first end of the template DNA molecule and a 3' end that hybridizes to a suitable location on the first end of the template DNA molecule;
- providing a second primer DNA molecule having a 5' end that comprises nucleotide sequences that will incorporate nucleotide sequences that are complementary to the second end of the linearized vector molecule onto the second end of the template DNA molecule and a 3' end that hybridizes to a suitable location on the second end of the template DNA molecule;
- amplifying the template DNA molecule using the polymerase chain reaction with the first and second primers to provide a PCR amplified product;
incubating the PCR amplified product with the linearized vector DNA
molecule in the presence of a DNA polymerase under conditions to generate a recombinant DNA molecule;
- transforming the recombinant DNA molecule into a host cell; and - isolating the recombinant DNA product;
wherein the DNA polymerase has intrinsic exonuclease activity and joins the DNA molecules.
31. A kit for direct cloning of a linear DNA molecule comprising, in separate containers, a DNA polymerase and a reaction buffer, wherein the DNA
polymerase has intrinsic exonuclease activity and is capable of performing a DNA joining reaction.
polymerase has intrinsic exonuclease activity and is capable of performing a DNA joining reaction.
32. The kit according to claim 31, further comprising a stimulatory factor.
33. The kit according to claim 32, wherein the stimulatory factor is a single strand DNA binding protein.
34. The kit according to any one of claims 31-33, further comprising reagents to perform a positive control reaction.
35. The kit according to claim 34, wherein the reagents to perform a positive control comprise a linearized vector, an insert DNA with first and second ends having appropriate complementary sequences, a DNA polymerase having intrinsic exonuclease activity that is capable of performing a DNA joining reaction, and a reaction buffer.
36. The kit according to claim 35, wherein the reagents to perform a positive control further comprise a stimulatory factor.
37. The kit according to claim 36, wherein the insert DNA is a PCR-amplified product.
38. A DNA molecule prepared according to the method of any one of claims 1 to 30.
39. The use of a DNA polymerase molecule having exonuclease activity and capable of joining DNA molecules, for cloning DNA molecules.
40. The use according to claim 39, wherein the DNA molecules are produced by PCR.
41. The use according to any of claims 39 or 40, wherein the DNA polymerase comprises a polymerase selected from the group consisting of vaccinia virus DNA polymerase, T4 DNA polymerase and the Klenow fragment of E. coli DNA polymerase I.
42. The use of the method according to claim 13 for site directed mutagenesis.
43. A composition for cloning DNA molecules, comprising a carrier and DNA
polymerase having exonuclease activity and capable of joining linear DNA
molecules.
polymerase having exonuclease activity and capable of joining linear DNA
molecules.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA2402321A CA2402321C (en) | 2000-03-07 | 2001-03-07 | Dna joining method |
Applications Claiming Priority (8)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US18740100P | 2000-03-07 | 2000-03-07 | |
| US60/187,401 | 2000-03-07 | ||
| CA2,317,865 | 2000-09-01 | ||
| CA002317865A CA2317865A1 (en) | 2000-03-07 | 2000-09-01 | Dna joining method |
| US26377101P | 2001-01-25 | 2001-01-25 | |
| US60/263,771 | 2001-01-25 | ||
| CA2402321A CA2402321C (en) | 2000-03-07 | 2001-03-07 | Dna joining method |
| PCT/CA2001/000283 WO2001066775A2 (en) | 2000-03-07 | 2001-03-07 | Dna joining method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2402321A1 true CA2402321A1 (en) | 2001-09-13 |
| CA2402321C CA2402321C (en) | 2012-12-18 |
Family
ID=27427641
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA2402321A Expired - Lifetime CA2402321C (en) | 2000-03-07 | 2001-03-07 | Dna joining method |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA2402321C (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112877324A (en) * | 2021-01-28 | 2021-06-01 | 杭州师范大学 | DNA cloning method |
| CN113667649A (en) * | 2021-09-08 | 2021-11-19 | 苏州博特龙免疫技术有限公司 | Reaction mixed reagent for lightning cloning and preparation method and application thereof |
| CN115948514A (en) * | 2022-11-10 | 2023-04-11 | 广州派真生物技术有限公司 | In-vitro amplification method of linear double-stranded DNA |
-
2001
- 2001-03-07 CA CA2402321A patent/CA2402321C/en not_active Expired - Lifetime
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112877324A (en) * | 2021-01-28 | 2021-06-01 | 杭州师范大学 | DNA cloning method |
| CN113667649A (en) * | 2021-09-08 | 2021-11-19 | 苏州博特龙免疫技术有限公司 | Reaction mixed reagent for lightning cloning and preparation method and application thereof |
| CN115948514A (en) * | 2022-11-10 | 2023-04-11 | 广州派真生物技术有限公司 | In-vitro amplification method of linear double-stranded DNA |
| CN115948514B (en) * | 2022-11-10 | 2023-09-12 | 广州派真生物技术有限公司 | In vitro amplification method of linear double-stranded DNA |
Also Published As
| Publication number | Publication date |
|---|---|
| CA2402321C (en) | 2012-12-18 |
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| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| MKEX | Expiry |
Effective date: 20210308 |